Pre-proglucagon is a 158 amino acid precursor polypeptide that is processed in different tissues to form a number of different proglucagon-derived peptides, including glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin (OXM), that are involved in a wide variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying, and intestinal growth, as well as the regulation of food intake. Glucagon is a 29-amino acid peptide that corresponds to amino acids 33 through 61 of pre-proglucagon, while GLP-1 is produced as a 37-amino acid peptide that corresponds to amino acids 72 through 108 of pre-proglucagon. GLP-1(7-36) amide or GLP-1(7-37) acid are biologically potent forms of GLP-1, that demonstrate essentially equivalent activity at the GLP-1 receptor.
Glucagon generally functions as a counter-regulatory hormone, opposing the actions of insulin, to maintain the level of blood glucose, particularly in instances of hypoglycemia. Thus, glucagon's general role in glucose regulation is to counteract the action of insulin and maintain blood glucose levels through enhanced synthesis and mobilization of glucose in the liver. However, in some patients with Type 1 or Type 2 diabetes, absolute or relative elevated glucagon levels have been shown to contribute to the hyperglycemic state. Both in healthy control animals as well as in animal models of Type 1 and Type 2 diabetes, removal of circulating glucagon with selective and specific antibodies has resulted in reduction of the glycemic level (Brand et al., Diabetologia 37, 985 (1994); Diabetes 43, [suppl 1], 172A (1994); Am. J. Physiol. 269, E469-E477 (1995); Diabetes 44 [suppl 1], 134A (1995); Diabetes 45, 1076 (1996)). These studies suggest that glucagon antagonism could be useful in glycemic control of diabetes.
Glucagon exerts its action by binding to and activating its receptor, which is part of the glucagon-secretin branch of the 7-transmembrane G-protein coupled receptor family. The receptor functions by an activation of the adenylyl cyclase resulting in increased cAMP levels. Previous reports have identified peptide-based, (see Unson, C. G. et al. (1989) J. Biol. Chem. 264, 789-94, Ahn, J. et al. (2001) J. Peptide Research 58, 151-8 and Ahn J. et al. (2001) J. Med. Chem. 44, 1372-9) as well as nucleotide-based glucagon antagonists (Sloop K. et al. (2004) J. Clinical Invest. 113, 1571-81). Peptide-based inhibition acts at the level of receptor binding while the latter functions by suppressing intracellular mRNA specific to the glucagon receptor.
Inhibitors of the glucagon receptor have been described, and are generally based on the amino acid sequence of glucagon. Several analogues in which one or more amino acids were either deleted or substituted to produce potent antagonists of glucagon receptor have been described, for example, [des His1] [Glu9]-glucagon amide (Unson et al., (1989) Peptides 10, 1171; Post et al., (1993) Proc. Natl. Acad. Sci. USA 90, 1662), des His1, Phe6 [Glu9]-glucagon amide (Azizh et al., (1995) Bioorg. & Med. Chem. Lett. 16, 1849) and Nle9, Ala11,16-glucagon amide (Unson et al. (1994) J. Biol. Chem. 269(17), 12548). Other analogues include substitutions at positions 4 (Ahn J M et al. (2001) J. Pept. Res. 58(2):151-8), 1 (Dharanipragada, R. et al. (1993) Int. J. Pept. Res. 42(1): 68-77) and 4, 5, 12, 17 and 18 in the glucagon sequence (Gysin B et al. 1986. Biochemistry. 25(25):8278-84).
GLP-1 has different biological activities compared to glucagon. Its actions include stimulation of insulin synthesis and secretion, inhibition of glucagon secretion, and inhibition of food intake. GLP-1 has been shown to reduce hyperglycemia (elevated glucose levels) in diabetics. Exendin-4, a peptide from lizard venom that shares about 50% amino acid identity with GLP-1, activates the GLP-1 receptor and likewise has been shown to reduce hyperglycemia in diabetics.
There is also evidence that GLP-1 and exendin-4 may reduce food intake and promote weight loss, an effect that would be beneficial not only for diabetics but also for patients suffering from obesity. Patients with obesity have a higher risk of diabetes, hypertension, hyperlipidemia, cardiovascular disease, and musculoskeletal diseases.
As described herein, glucagon analogs are provided that exhibit high potency activity, both as glucagon antagonists and as GLP-1 agonists. More particularly, the novel glucagon antagonist/GLP-1 agonists represent novel chemical modifications of the N-terminus of the native glucagon sequence, and substitutions of the native glucagon sequence that allow for stabilization of the alpha-helix structure in the C-terminal portion of the compound. The novel glucagon antagonist/GLP-1 agonist compounds can be used in any setting where glucagon antagonism concurrent with GLP-1 agonism is desired. In accordance with one embodiment the compounds can be used in the treatment of diabetes or obesity.